Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics

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Abstract
The ability to edit a specific gene within our genomes using guided-nucleases (Cas9/ZFN/TALEN - CaZiTa) presents huge opportunities for curing many genetic disorders. Delivery of this ‘drug’ within cells is a critical step for such therapies. The ability of recombinant adeno-associated virus (rAAV) to enter cells makes it a perfect choice as a vector for gene therapy. A plasmid comprising the rAAV, the CaZiTa, guide RNAs (for CRISPR) is expected to enter the cell, edit the target gene(s), remain episomal, and thus fade away with time. However, the rather obvious danger of integration of the plasmid into the genome, if the episomal hypothesis is incorrect, is under-reported. A recent report has highlighted that bacterial genes from a plasmid were integrated into bovine genomes. Massachusetts General Hospital has recently published data on CRISPR edits (Accid:PRJNA563918), noting ‘high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks’. However, there is no mention of Cas9 integration. Here, the same data from Massachusetts General Hospital shows Cas9 integration in the exact edit sites provided for two genes - TMC1 and DMD. Also, there is a mis-annotation of one sample as ‘no gRNA’, since Cas9 integrations have been detected in that sample. This is an important distinction between AAV and CaZiTa integration: while AAV integration can be tolerated, Cas9 integration is a huge, and unacceptable, danger.

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